Renin inhibitory peptides having His13

ABSTRACT

Renin inhibitory peptides of the formula ##STR1## and analogs thereof inhibit renin and are useful for treating various forms of renin-associated hypertension and hyperaldosteronism.

REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of application Ser. No. 312,558, filedOct. 19, 1981, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is concerned with novel peptides which inhibitrenin.

The present invention is also concerned with pharmaceutical compositionscontaining the novel peptides of the present invention as activeingredients, with methods of treating renin-associated hypertension andhyperaldosteronism, with diagnostic methods which utilize the novelpeptides of the present invention, and with methods of preparing thenovel peptides of the present invention.

Renin is a proteolytic enzyme of molecular weight about 40,000, producedand secreted by the kidney. It is secreted by the juxtaglomerular cellsand acts on the plasma substrate, angiotensinogen, to split off thedecapeptide angiotensin I, which is converted to the potent pressoragent angiotensin II. Thus, the renin-angiotensin system plays animportant role in normal cardiovascular homeostasis and in some forms ofhypertension.

In the past, attempts to modulate or manipulate the renin-angiotensinsystem have met with success in the use of inhibitors of angiotensin Iconverting enzyme. In view of this success, it seems reasonable toconclude that a specific inhibitor of the limiting enzymatic step thatultimately regulates angiotensin II production, the action of renin onits substrate, would be at least equally successful. Thus, an effectiveinhibitor of renin has been long sought as both a therapeutic agent andas an investigative tool.

2. Brief Description of the Prior Art

There has been substantial interest in the synthesis of useful renininhibitors for many decades; and the following table lists the majorclasses of renin inhibitors that have been studied, as well as theirinhibition constants (K_(i)):

    ______________________________________                                        Class             K.sub.i (M)                                                 ______________________________________                                        Renin antibody    probably 10.sup.-6                                          Pepstatin         10.sup.-6 - 10.sup.-7                                       Phospholipids     10.sup.-3                                                   Substrate analogs                                                             Tetrapeptides     10.sup.-3                                                   Octa- to tridecapeptides                                                                        10.sup.-5 - 10.sup.-6                                       ______________________________________                                    

Umezawa et al., in J. Antibiot. (Tokyo) 23: 259-262, 1970, reported theisolation of a peptide from actinomyces that was an inhibitor ofaspartyl proteases such as pepsin, cathepsin D, and renin. This peptide,known as pepstatin, was found by Gross et al., Science 175:656, 1971, toreduce blood pressure in vivo after the injection of hog renin intonephrectomized rats. However, pepstatin has not found wide applicationas an experimental agent because of its limited solubility and itsinhibition of a variety of other acid proteases in addition to renin.The structure of pepstatin is shown below: ##STR2##

To date, many efforts have been made to prepare a specific renininhibitor based on substrate analogy. Since the human renin substratehas only recently been elucidated (Tewksbury et al., Circulation 59, 60,Supp. II: 132, Oct. 1979), heretofore substrate analogy has been basedon the known pig renin substrate. While the human and pig reninsubstrates are not the same, the substrate analogy based on pig reninhas always been considered acceptable in the art as predictive of humanrenin inhibitory activity because of the closely related activity of thetwo renins. Thus, while pig renin does not cleave the human reninsubstrate, human renin, on the other hand, does cleave the pig reninsubstrate. See Poulsen et al., Biochim. Biophys. Acta 452:533-537, 1976;and Skeggs, Jr. et al., J. Exp. Med. 106:439-453, 1957.

It has been found, for example, using pig renin substrate analogy, thatthe octapeptide sequence extending from histidine-6 through tyrosine-13has kinetic parameters essentially the same as those of the fulltetradecapeptide renin substrate. The amino acid sequence of theoctapeptide in pig renin substrate is as follows: ##STR3## Renin cleavesthis substrate between Leu¹⁰ and Leu¹¹.

The renin inhibitory peptides of the present invention are based onhuman renin substrate analogy. The human renin octapeptide sequence,related to the pig renin substrate octapeptide, is as follows: ##STR4##Similar to the pig renin substrate, human renin cleaves this substratebetween Leu¹⁰ and Val¹¹.

Kokubu et al., Biochem. Pharmacol. 22: 3217-3223, 1973, synthesized anumber of analogs of the tetrapeptide found between residues 10 to 13 ofthe pig renin substrate, but while inhibition could be shown, inhibitoryconstants were only of the order of 10⁻³ M.

Analogs of a larger segment of renin substrate were also synthesized:Burton et al., Biochemistry 14: 3892-3898, 1975, and Poulsen et al.,Biochemistry 12: 3877-3882, 1973. Two of the major obstacles which hadto be overcome to obtain an effective renin inhibitor useful in vivowere lack of solubility and weak binding (large inhibitory constant).Modifications to increase solubility soon established that theinhibitory properties of the peptides are markedly dependent on thehydrophobicity of various amino acid residues, and that increasingsolubility by replacing lipophilic amino acids with hydrophilicisosteric residues becomes counter-productive. Other approaches toincreasing solubility have had limited success. Various modificationsdesigned to increase binding to renin have also been made, but here too,with only limited success. For a more detailed description of pastefforts to prepare an effective inhibitor of renin, see Haber andBurton, Fed. Proc. Fed. Am. Soc. Exp. Biol. 38:2768-2773, 1979.

For other articles describing previous efforts to devise renininhibitors, see Marshall, Federation Proc. 35: 2494-2501, 1976; Burtonet al., Proc. Natl. Acad. Sci. USA 77: 5476-5479, Sept. 1980; Suketa etal., Biochemistry 14: 3188, 1975; Swales, Pharmac. Ther. 7: 173-201,1979; Kokubu et al., Nature 217: 456-457, Feb. 3, 1968; Matsushita etal., J. Antibiotics 28: 1016-1018, Dec. 1975; Lazar et al., Biochem.Pharma. 23: 2776-2778, 1974; Miller et al., Biohem. Pharma. 21:2941-2944, 1972; Haber, Clinical Science 59: 7s-19s, 1980; and Rich etal., J. Org. Chem. 43: 3624, 1978, and J. Med. Chem. 23: 27, 1980.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

In accordance with the present invention there are provided renininhibitory peptides of the formula: ##STR5## wherein: A is hydrogen;##STR6## where n is 0 to 5 and R³ has the same meaning as set outfurther below, and may additionally be hydrogen;

B is absent; glycyl; sarcosyl; or ##STR7## D is absent; or ##STR8##where Z is (CH₂)_(n) and n is 1 or 2; or --S--; R¹ is hydrogen; C₁₋₄alkyl; hydroxy C₁₋₄ alkyl; phenyl; phenyl mono-substituted with a memberselected from the group consisting of methyl, trifluoromethyl, hydroxy,methoxy, fluoro, chloro, bromo, and iodo; indolyl; 4-imidazolyl; amineC₂₋₄ alkyl; guanidyl C₂₋₃ alkyl; or methylthiomethyl;

R² is hydrogen C₁₋₄ ; alkyl; phenyl; phenyl mono-substituted with amember selected from the group consisting of methyl, trifluoromethyl,hydroxy, methoxy, fluoro, chloro, bromo, and iodo; or indolyl;

R³ is C₃₋₆ alkyl; C₃₋₇ cycloalkyl; phenyl; or phenyl mono-substitutedwith a member selected from the group consisting of methyl,trifluoromethyl, hydroxy, methoxy, fluoro, chloro, bromo, and iodo;

R⁴ is hydrogen; or ##STR9## where R' is hydrogen; C₁₋₄ alkyl; hydroxy;or C₃₋₇ cycloalkyl; R⁵ is 4-imidazolyl; amino C₂₋₄ alkyl; 2-, 3-, or4-pyridyl; or guanidyl C₂₋₃ alkyl; and

E is OR"; NHR", or N(R")₂, where each R" may be the same or differentand is hydrogen or C₁₋₄ alkyl;

wherein all of the asymmetric carbon atoms have an S configuration,except those of substituents B and D, which may also have the Rconfiguration; and a pharmaceutically acceptable salt thereof.

In the above definitions, the term "alkyl" is intended to include bothbranched and straight chain hydrocarbon groups having the indicatednumber of carbon atoms.

The novel renin inhibitory peptides of the present invention may also bedescribed in terms of common amino acid components and closely relatedanalogs thereof, in accordance with the following formula:

    A-B-B-D-F-G-Sta-H-I-B-E                                    (II)

The A,B,D, and E components correspond to the same portions of FormulaI.

In Formula II, Sta represents the unusual amino acid statine and itsclosely related analogs, and its presence constitutes a unique featureof the renin inhibitory peptides of the present invention. Statine maybe named as 4(S)-amino-3(S)-hydroxy-6-methylheptanoic acid, and may berepresented by the following formula: ##STR10## As shown in Formula IIIabove, the delta-substituent in naturally-occurring statine isisopropyl, or a leucine sidechain, essentially. As shown in Formula I bythe R³ substituents, the isopropyl group may be replaced by higher alkylgroups up to six carbon atoms, cycloalkyl groups containing from threeto seven carbon atoms, phenyl, and phenyl monosubstituted with a memberselected from the group consisting of methyl, trifluoromethyl, hydroxy,methoxy, fluoro, chloro, bromo, and iodo. The phenyl substituent isespecially preferred. These modifications of the naturally-occurringstatine structure are in accordance with the hydrophobicity considerednecessary to maintain the inhibitory activity of the total peptide.

The remaining common amino acid components of Formula II are as follows:

A has the same meaning as above in Formula I;

B is absent, Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, His, Lys, Orn, Arg,or Met;

D is absent or Pro;

F is Ala, Leu, Phe, Tyr, or Trp;

G is Ala, Leu, Phe, Tyr, Trp, His, Lys, Orn, Arg, or Met;

H is the same as F and may additionally be Ser, Gly, Val, Ile, or Thr;

I is His, Arg, Lys, or Orn; and

E has the same meaning as above in Formula I.

It will be understood that closely related analogs of the above commonamino acids, for example, aliphatic amino acids in addition to Ala, Val,Leu, and Ile, such as α-aminobutyric acid (Abu), and substituted phenylderivatives of Phe, are included in the broad description of the novelinhibitory peptides of the present invention represented by Formula Iand its definitions. Thus, the peptides of Formula II and itsdefinitions, including the derivatives of naturally-occurring statinerepresented by the definitions of the R³ substituent in Formula I,represent preferred peptides of the present invention.

Especially preferred inhibitory peptides of the present invention arethe following:

iso-Butyryl-His-Pro-Phe-His-Sta-Val-His-Gly-NH₂

iso-Butyryl-His-Pro-Phe-His-Sta-Ile-His-NH₂

tert-Butyloxycarbonyl-Phe-His-Sta-Ile-His-NH₂

Benzyloxycarbonyl-Phe-His-Sta-Ile-His-NH₂

iso-Valeryl-His-Pro-Phe-His-Sta-Ile-His-NH₂

iso-Valeryl-His-Pro-Phe-His-Sta-Leu-His-NH₂

The inhibitory peptides of the present invention may be betterappreciated in terms of substrate analogy from the followingillustration of Formula I alongside the octapeptide sequence of aportion of the human renin substrate, which renin cleaves between Leu¹⁰and Val¹¹ :

    ______________________________________                                         ##STR11##                                                                

    ______________________________________                                         ##STR12##                    (IV)                                            ______________________________________                                    

As can be seen, a unique aspect and essential feature of the presentinvention is the substitution of the single statine amino acid componentfor the double amino acid sequence: Leu¹⁰ -Val¹¹ in the endogenous humanrenin substrate. It is believed that substitution of statine for boththe leucine and valine amino acids rather than just the leucine resultsin an improved substrate analogy due to the greater linear extent ofstatine as compared to a single leucine component. Thus, statine moreclosely approximates Leu-Val in linear extent, and thereby provides abetter "fit" to the human renin enzyme.

The Formula I compounds can be used in the form of salts derived frominorganic or organic acids and bases. Included among such acid additionsalts are the following: acetate, adipate, alginate, aspartate,benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate,camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate.Base salts include ammonium salts, alkali metal salts such as sodium andpotassium salts, alkaline earth metal salts such as calcium andmagnesium salts, salts with organic bases such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such asarginine, lysine, and so forth. Also, the basic nitrogen-containinggroups can be quaternized with such agents as lower alkyl halides, suchas methyl, ethyl, propyl, and butyl chloride, bromides and iodides;dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates,long chain halides such as decyl, lauryl, myristyl and stearylchlorides, bromides and iodides, aralkyl halides like benzyl andphenethyl bromides and others. Water or oil-soluble or dispersibleproducts are thereby obtained.

The novel peptides of the present invention possess an excellent degreeof activity in treating renin-associated hypertension andhyperaldosteronism.

For these purposes the compounds of the present invention may beadministered parenterally, by inhalation spray, or rectally in dosageunit formulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and vehicles. The term parenteral as usedherein includes subcutaneous injections, intravenous, intramuscular,intrasternal injection or infusion techniques. In addition to thetreatment of warm-blooded animals such as mice, rats, horses, dogs,cats, etc., the compounds of the invention are effective in thetreatment of humans.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example as a sterile injectable aqueous oroleagenous suspension. This suspension may be formulated according tothe known art using suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation may also be a sterileinjectable solution or suspension in a non-toxic parenterally-acceptablediluent or solvent, for example as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectibles.

The peptides of this invention may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials are cocoa butter and polyethylene glycols.

Dosage levels of the order of 2 to 35 grams per day are useful in thetreatment of the above indicated conditions. For example,renin-associated hypertension and hyperaldosteronism are effectivelytreated by the administration of from 30 milligrams to 0.5 grams of thecompound per kilogram of body weight per day.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy.

Thus, in accordance with the present invention there is further provideda pharmaceutical composition for treating renin-associated hypertensionand hyperaldosteronism, comprising a pharmaceutical carrier and atherapeutically effective amount of a peptide of the formula: ##STR13##wherein A, B, D, R¹, R², R³, R⁴, R⁵, and E have the same meaning asrecited further above for Formula I; wherein all of the asymmetriccarbon atoms have an S configuration, except those of substituents B andD, which may also have the R configuration; and a pharmaceuticallyacceptable salt thereof.

Also, in accordance with the present invention there is still furtherprovided a method of treating renin-associated hypertension andhyperaldosteronism, comprising administering to a patient in need ofsuch treatment, a therapeutically effective amount of a peptide of theformula: ##STR14## wherein A, B, D, R¹, R², R³, R⁴, R⁵, and E have thesame meaning as recited further above for Formula I; wherein all of theasymmetric carbon atoms have an S configuration, except those ofsubstituents B and D, which may also have the R configuration; and apharmaceutically acceptable salt thereof.

The renin inhibitory novel peptides of the present invention may also beutilized in diagnostic methods for the purpose of establishing thesignificance of renin as a causative or contributory factor inhypertension or hyperaldosteronism in a particular patient. For thispurpose the novel peptides of the present invention may be administeredin a single dose of from 0.1 to 10 mg per kg of body weight.

Both in vivo and in vitro methods may be employed. In the in vivomethod, a novel peptide of the present invention is administered to apatient, preferably by intravenous injection, although parenteraladministration is also suitable, at a hypotensive dosage level and as asingle dose, and there may result a transitory fall in blood pressure.This fall in blood pressure, if it occurs, indicates supranormal plasmarenin levels.

An in vitro method which may be employed involves incubating a bodyfluid, preferably plasma, with a novel peptide of the present inventionand, after deproteinization, measuring the amount of angiotensin IIproduced in nephrectomized, pentolinium-treated rats. Another in vitromethod involves mixing the plasma or other body fluid with a novelpeptide of the present invention and injecting the mixture into a testanimal. The difference in pressor response with and without addedpeptide is a measure of the renin content of the plasma.

Pepstatin may be employed in the methods described above as an activecontrol. See, e.g., U.S. Pat. Nos. 3,784,686 and 3,873,681 for adescription of the use of pepstatin in diagnostic methods of this type.

The novel peptides of the present invention may be prepared inaccordance with well-known procedures for preparing peptides from theirconstituent amino acids, which will be described in more detail below.The unusual amino acid, statine, may be prepared in accordance with theprocedure described in Rich et. al., J. Org. Chem. 43:3624 (1978).

The novel inhibitory peptides of the present invention are prepared byusing the solid phase sequential synthesis technique.

In the following description several abbreviated designations are usedfor the amino acid components, certain preferred protecting groups,reagents and solvents. The meanings of such abbreviated designations aregiven below in Table I.

                  TABLE I                                                         ______________________________________                                        Abbreviated                                                                   Designation                                                                   ______________________________________                                                      Amino Acid                                                      Ala           L-alanine                                                       Arg           L-arginine                                                      Gly           L-glycine -His D or L-histidine                                 Ile           L-isoleucine                                                    Leu           L-leucine                                                       Lys           L-lysine                                                        Met           L-methionine                                                    Orn           L-ornithine                                                     Phe           L-phenylalanine                                                 Pro           D or L-proline                                                  Sar           L-sarcosine (N--methylglycine)                                  Ser           L-serine                                                        Sta           (3S,4S)-statine                                                 Thr           L-threonine                                                     Trp           L-tryptophan                                                    Tyr           L-tyrosine                                                      Val           L-valine                                                                       Protecting Groups                                              BOC           tert-butyloxycarbonyl                                           CBZ           benzyloxycarbonyl                                               DNP           dinitrophenyl                                                   OMe           methyl ester                                                                  Activating Groups                                               HBT           1-hydroxybenzotriazole                                                        Condensing Agents                                               DCCI          dicyclohexylcarbodiimide                                        DPPA          diphenylphosphorylazide                                                       Reagents                                                        TEA           triethylamine                                                   TFA           trifluoroacetic acid                                                          Solvents                                                        A             ammonium hydroxide (conc.)                                      AcOH          acetic acid                                                     C             chloroform                                                      DMF           dimethylformamide                                               E             ethyl acetate                                                   M             methanol                                                        P             pyridine                                                        THF           tetrahydrofuran                                                 W             water                                                           ______________________________________                                    

The synthesis of the peptides of the present invention by the solidphase technique is conducted in a stepwise manner on chloromethylatedresin. The resin is composed of fine beads (20-70 microns in diameter)of a synthetic resin prepared by copolymerization of styrene with 1-2percent divinylbenzene. The benzene rings in the resin arechloromethylated in a Friedel-Crafts reaction with chloromethyl methylether and stannic chloride. The Friedel-Crafts reaction is continueduntil the resin contains 0.5 to 5 mmoles of chlorine per gram of resin.

The amino acid selected to be the C-terminal amino acid of the linearpeptide is converted to its amino protected derivative. The carboxylgroup of the selected C-terminal amino acid is bound covalently to theinsoluble polymeric resin support, as for example, as the carboxylicester of the resin-bonded benzyl chloride present inchloromethyl-substituted polystyrene-divinylbenzene resin. After theamino protecting group is removed, the amino protected derivative of thenext amino acid in the sequence is added along with a coupling agent,such as dicyclohexylcarbodiimide. The amino acid reactant may beemployed in the form of a carboxyl-activated amino acid such as ONpester, an amino acid azide, and the like. Deprotection and addition ofsuccessive amino acids is performed until the desired linear peptide isformed.

The selection of protecting groups is, in part, dictated by particularcoupling conditions, in part by the amino acid and peptide componentsinvolved in the reaction.

Amino-protecting groups ordinarily employed include those which are wellknown in the art, for example, urethane protecting substituents such asbenzyloxy-carbonyl (carbobenzoxy), p-methoxycarbobenzoxy,p-nitrocarbobenzoxy, t-butyoxycarbonyl, and the like. It is preferred toutilize t-butyloxycarbonyl (BOC) for protecting the α-amino group in theamino acids undergoing reaction at the carboxyl end of said amino acid.The BOC protecting group is readily removed following such couplingreaction and prior to the subsequent step by the relatively mild actionof acids (i.e. trifluoroacetic acid, or hydrogen chloride in ethylacetate).

The OH group of Thr and Ser can be protected by the Bzl group and theα-amino group of Lys can be protected by the INOC group or the2-chlorobenzyloxycarbonyl (2-CL-CBZ) group. Neither group is affected byTFA, used for removing BOC protecting groups. After the peptide isformed, the protective groups, such as 2-Cl-CBZ and Bzl, can be removedby treatment with HF or by catalytic hydrogenation.

After the peptide has been formed on the solid phase resin, it may beremoved from the resin by a variety of methods which are well known inthe art. For example the peptide may be cleaved from the resin withhydrazine, by ammonia in methanol, or by methanol plus a suitable base.

Preparation of the novel inhibitory peptides of the present inventionutilizing the solid phase technique is illustrated in the followingexamples. These examples are predictive and have not actually beencarried out. Characterization data is based on an actual preparationinvolving a method less facile than the solid phase technique describedin the examples. The hog renin and human renin inhibition assays wereactually carried out.

EXAMPLE 1N-Isobutyryl-L-histidyl-L-prolyl-L-phenylalanyl-L-histidyl-(3S,4S)-statyl-L-valyl-L-histidyl-L-glycylamide

The title peptide is prepared by standard solid phase methodology, asdescribed in Erickson and Merrifield, Proteins, 3rd et., 2:257-527,1976, using a Beckman Model 990B peptide synthesizer to carry out theoperations according to the attached programs. The starting polymer isBOC-Gly esterified to 2% cross-linked polystyrene-divinylbenzene (6mmol, 5.00 g). The N.sup.α -BOC derivatives of His-DNP, Val, Sta,His-DNP, Phe, and Pro are coupled using dicyclohexylcarbodiimide with anequivalent of the additive 1-hydroxybenzotriazole hydrate. The Sta isprepared in accordance with Rich et al., J. Org. Chem. 43:3624, 1978.The BOC-group is removed with 40% trifluoroacetic acid. A coupling of 60minutes followed by a recoupling of 120 minutes (2.5 equivalents eachtime of BOC-amino acid) are used for each amino acid, except for Sta.These coupling times have been previously demonstrated to give completecoupling (as judged by the method of Kaiser) in this sequence. In orderto conserve the amounts of Sta employed, an initial coupling using 1.08equivalents of N.sup.α -BOC-Sta (in 20 ml of 1:1 CH₂ Cl₂ /DMF) for 72hrs gives approximately 95% complete reaction. The addition of anadditional 0.12 equivalents of N.sup.α -BOC-Sta plus an equal amount ofDCCI to the stirring suspension gives complete coupling after anadditional 18 hrs. The N-terminal isobutyryl group is coupled for 60minutes as the symmetrical anhydride generated in situ from 5.0equivalents of isobutyric acid and 2.5 equivalents of DCCI. This isfollowed by a recoupling for 120 minutes using 2.5 equivalents ofisobutyric acid, HBT, and DCCI. The DNP protecting groups on His areremoved in the final program using two 25-minute treatments with 10%thiophenol in DMF. The finished resin-peptide is dried and suspended in30 ml methanol containing 1 g ammonium acetate in a 100 ml pressurebottle containing a magnetic stirring bar. The suspension is cooledunder nitrogen to -20° C., and anhydrous ammonia is bubbled through thesuspension until it is saturated. The pressure bottle is then closed andallowed to warm to room temperature. The suspension is stirred for 72hrs, after which the pressure valve is carefully opened, allowing theammonia to escape. The suspension of beads in orange-red solution isfiltered and the beads washed. The filtrate and washes are evaporatedand the solid dried. This crude product is then partitioned betweenwater (200 ml) and ethyl acetate (200 ml). The ethyl acetate layer iswashed three times with 100 ml of a 1% citric acid solution. The productand the acid layer are neutralized with solid sodium hydrogencarbonate.A yellow oil precipitates from the water when it becomes basic. The nowbasic water solution and precipitated oil are extracted four times with100 ml of dichloromethane, and the organic layers are dried andevaporated to give 6.4 g of crude yellow solid. This solid is dissolvedin 50 ml of 80:20:2.5, chloroform/methanol/water and loaded onto asilica column (E. Merck No. 9385 silica, 0.040-0.063 mm particle size)8.9×47 cm (ca. 1500 g silica gel) which has been packed in the samesolvent. The column is eluted at 18 ml/min with the same solvent and,after 2700 ml, fractions are collected (27 ml each). The pure product isidentified by TLC of the fractions. These fractions are combined andevaporated to a light yellow oil. The oil is dissolved in 300 ml ofwater. The solution is filtered (10μ) and freeze dried to give the finalproduct.

    ______________________________________                                        TLC:    50:40:10     C/M/A    Rf = 0.66                                       ______________________________________                                    

For all of the above no impurities are detected at the 2% level, i.e.,the product is greater than 99% pure.

HPLC: Greater than 99% single peak.

    ______________________________________                                               Spinco:                                                                             His    2.02                                                                   Pro    1.00                                                                   Phe    0.99                                                                   Val    0.99                                                                   Gly    0.99                                                      ______________________________________                                    

SCHEDULE OF STEPS FOR 6 MMOL RUN

    ______________________________________                                        Step Solvent/Reagent  Vol. (ml)                                                                              Mix time (min)                                 ______________________________________                                        Coupling Program 1                                                            1    CH.sub.2 Cl.sub.2                                                                              6 × 60                                                                           2                                              2    40% TFA in CH.sub.2 Cl.sub.2                                                                   1 × 60                                                                           2                                              3    40% TFA in CH.sub.2 Cl.sub.2                                                                   1 × 60                                                                           25                                             4    CH.sub.2 Cl.sub.2                                                                              3 × 60                                                                           2                                              5    10% TEA in CH.sub.2 Cl.sub.2                                                                   2 × 60                                                                           5                                              6    CH.sub.2 Cl.sub.2                                                                              3 × 60                                                                           2                                              7    BOC-amino acid, HBT                                                                            40       5                                                   in 1:1 DMF/CH.sub.2 Cl.sub.2                                             8    1.0M DCCI in CH.sub.2 Cl.sub.2                                                                 15       60                                             9    DMF              1 × 60                                                                           2                                              10   MeOH             2 × 60                                                                           2                                              11   CH.sub.2 Cl.sub.2                                                                              1 × 60                                                                           2                                              Re-Couple Program 2                                                           1    CH.sub.2 Cl.sub.2                                                                              1 × 60                                                                           2                                              2    10% TEA in CH.sub.2 Cl.sub.2                                                                   2 × 60                                                                           5                                              3    CH.sub.2 Cl.sub.2                                                                              3 × 60                                                                           2                                              4    BOC-amino acid, HBT                                                                            40       5                                                   in 1:1 DMF/CH.sub.2 Cl.sub.2                                             5    1.0M DCCI in CH.sub.2 Cl.sub.2                                                                 15       120                                            6    DMF              1 × 60                                                                           2                                              7    MeOH             2 × 60                                                                           2                                              8    CH.sub.2 Cl.sub.2                                                                              5 × 60                                                                           2                                              Program 3 (DNP removal)                                                       1    CH.sub.2 Cl.sub.2                                                                              1 × 60                                            2    DMF              2 × 60                                                                           2                                              3    10% phenylthiol in DMF                                                                         1 × 60                                                                           25                                             4    DMF              1 × 60                                                                           2                                              5    10% TEA in CH.sub.2 Cl.sub.2                                                                   1 × 60                                                                           2                                              6    DMF              2 × 60                                                                           2                                              7    10% phenylthiol in DMF                                                                         1 × 60                                                                           25                                             8    DMF              3 × 60                                                                           2                                              9    MeOH             2 × 60                                                                           2                                              10   CH.sub.2 Cl.sub.2                                                                              2 × 60                                                                           2                                              11   MeOH             2 × 60                                                                           2                                              12   CH.sub.2 Cl.sub.2                                                                              2 × 60                                                                           2                                              13   MeOH             2 × 60                                                                           2                                              ______________________________________                                    

EXAMPLE 2-6

Following the standard solid phase methodology described above inExample 1, additional inhibitory peptides of the present invention areprepared, substituting equivalent amounts of the appropriate BOC-aminoacid for those utilized in Example 1, and, where necessary, providingN-terminal groups as substitutes for the isobutyryl group in accordancewith well established procedures in the art. The peptides prepared areset out in the following table.

    ______________________________________                                        Example Peptide                                                               No.     (5)  6  7   8  9 10(11) 12  13  (14)                                  ______________________________________                                        2       IBU*--His--Pro--Phe--His--Sta--Ile--His--NH.sub.2                     3       BOC--Phe--His--Sta--Ile--His--NH.sub.2                                4       CBZ--Phe--His--Sta--Ile--His--NH.sub.2                                5       IVA**--His--Pro--Phe--His--Sta--Ile--His--NH.sub.2                    6       IVA--His--Pro--Phe--His--Sta--Leu--His--NH.sub.2                      ______________________________________                                         *IBU = isobutyryl                                                             **IVA = isovaleryl                                                       

EXAMPLE 7 Pig Renin and Human Renin Inhibition

In order to illustrate the pig renin inhibition and human renininhibition of the peptide inhibitor of Example 1, the peptide wasevaluated in the pig renin inhibition assay described in Rich et al., J.Med. Chem. 23:27, 1980, and was further evaluated in the human reninassay based on the method of Haber et al., J. Clin. Endocrinol. 29:1349,1969. The latter method employs a radioimmunoassay technique to measurethe amount of angiotensin I product created by renin cleavage of itssubstrate. Human plasma (lyophilized) was used as the source of humansubstrate and human renin. I₅₀ values were obtained by plotting data atseveral inhibitor concentrations. The comparative results areillustrated below. Pepstatin was used as an active control.

    ______________________________________                                                         I.sub.50 (M)                                                 Peptide            Pig renin Human renin                                      ______________________________________                                        iso-Butyryl-His-- Pro--Phe--His--                                                                5.4 × 10.sup.-7                                                                   7.3 × 10.sup.-8                            Sta--Val--His--Gly--NH.sub.2                                                  iso-Valeryl-His-- Pro--Phe--His--                                                                9.3 × 10.sup.-7                                                                   7.3 × 10.sup.-9                            Sta--Ile--His--NH.sub.2                                                       Pepstatin(iso-valeryl-Val--                                                                      1.0 × 10.sup.-6                                                                   >>1.0 × 10.sup.-6                          Val--Sta--Ala--Sta)          (20% inhibition                                                               at 1.0 × 10.sup.-6)                        ______________________________________                                    

What is claimed is:
 1. A peptide of the formula: ##STR15## wherein: A ishydrogen; ##STR16## where n is 0 to 5 and R³ has the same meaning as setout further below, and may additionally be hydrogen;B is absent; glycyl;sarcosyl; or ##STR17## D is absent; or ##STR18## where Z is (CH₂)_(n)and n is 1 or 2; or --S--; R¹ is hydrogen; C₁₋₄ alkyl; hydroxy C₁₋₄alkyl; phenyl; phenyl mono-substituted with a member selected from thegroup consisting of methyl, trifluoromethyl, hydroxy, methoxy, fluoro,chloro, bromo, and iodo; indolyl; 4-imidazolyl; amine C₂₋₄ alkyl;guanidyl C₂₋₃ alkyl; or methylthiomethyl; R² is hydrogen C₁₋₄ ; alkyl;phenyl; phenyl mono-substituted with a member selected from the groupconsisting of methyl, trifluoromethyl, hydroxy, methoxy, fluoro, chloro,bromo, and iodo; or indolyl; R³ is C₃₋₆ alkyl; C₃₋₇ cycloalkyl; phenyl;or phenyl mono-substituted with a member selected from the groupconsisting of methyl, trifluoromethyl, hydroxy, methoxy, fluoro, chloro,bromo, and iodo; R⁴ is hydrogen; or ##STR19## where R' is hydrogen; C₁₋₄alkyl; hydroxy; or C₃₋₇ cycloalkyl; R⁵ is 4-imidazolyl; amino C₂₋₄alkyl; 2-, 3-, or 4-pyridyl; or guanidyl C₂₋₃ alkyl; and E is OR"; NHR",or N(R")₂, where each R" may be the same or different and is hydrogen orC₁₋₄ alkyl;wherein all of the asymmetric carbon atoms have an Sconfiguration, except those of substituents B and D, which may also havethe R configuration; and a pharmaceutically acceptable salt thereof. 2.A peptide according to claim 1 wherein the peptide isiso-butyryl-His-Pro-Phe-His-Sta-Val-His-Gly-NH₂.
 3. A peptide accordingto claim 1 wherein the peptide isiso-butyryl-His-Pro-Phe-His-Sta-Ile-His-NH₂.
 4. A peptide according toclaim 1 wherein the peptide istert-butyloxycarbonyl-Phe-His-Sta-Ile-His-NH₂.
 5. A peptide according toclaim 1 wherein the peptide isbenzyloxycarbonyl-Phe-His-Sta-Ile-His-NH₂.
 6. A peptide according toclaim 1 wherein the peptide isiso-valeryl-His-Pro-Phe-His-Sta-Ile-His-NH₂.
 7. A peptide according toclaim 1 wherein the peptide isiso-valeryl-His-Pro-Phe-His-Sta-Leu-His-NH₂.
 8. A pharmaceuticalcomposition for treating renin-associated hypertension, comprising apharmaceutical carrier and a therapeutically effective amount of apeptide of the formula: ##STR20## wherein: A is hydrogen; ##STR21##where n is 0 to 5 and R³ has the same meaning as set out further below,and may additionally be hydrogen;B is absent; glycyl; sarcosyl; or##STR22## D is absent; or ##STR23## where Z is (CH₂)_(n) and n is 1 or2; or --S--; R¹ is hydrogen; C₁₋₄ alkyl; hydroxy C₁₋₄ alkyl; phenyl;phenyl mono-substituted with a member selected from the group consistingof methyl, trifluoromethyl, hydroxy, methoxy, fluoro, chloro, bromo, andiodo; indolyl; 4-imidazolyl; amine C₂₋₄ alkyl; guanidyl C₂₋₃ alkyl; ormethylthiomethyl; R² is hydrogen C₁₋₄ ; alkyl; phenyl; phenylmono-substituted with a member selected from the group consisting ofmethyl, trifluoromethyl, hydroxy, methoxy, fluoro, chloro, bromo, andiodo; or indolyl; R³ is C₃₋₆ alkyl; C₃₋₇ cycloalkyl; phenyl; or phenylmono-substituted with a member selected from the group consisting ofmethyl, trifluoromethyl, hydroxy, methoxy, fluoro, chloro, bromo, andiodo; R⁴ is hydrogen; or ##STR24## where R' is hydrogen; C₁₋₄ alkyl;hydroxy; or C₃₋₇ cycloalkyl; R⁵ is 4-imidazolyl; amino C₂₋₄ alkyl; 2-,3-, 4-pyridyl; or guanidyl C₂₋₃ alkyl; and E is OR"; NHR", or N(R")₂,where each R" may be the same or different and is hydrogen or C₁₋₄alkyl;wherein all of the asymmetric carbon atoms have an Sconfiguration, except those of substituents B and D, which may also havethe R configuration; and a pharmaceutically acceptable salt thereof. 9.A method of treating renin-associated hypertension comprisingadministering to a patient in need of such treatment, a therapeuticallyeffective amount of a peptide of the formula: ##STR25## wherein: A ishydrogen; ##STR26## where n is 0 to 5 and R³ has the same meaning as setout further below, and may additionally be hydrogen;B is absent; glycyl;sarcosyl; or ##STR27## D is absent; or ##STR28## where Z is (CH₂)_(n)and n is 1 or 2; or --S--; R¹ is hydrogen; C₁₋₄ alkyl; hydroxy C₁₋₄alkyl; phenyl; phenyl mono-substituted with a member selected from thegroup consisting of methyl, trifluoromethyl, hydroxy, methoxy, fluoro,chloro, bromo, and iodo; indolyl; 4-imidazolyl; amine C₂₋₄ alkyl;guanidyl C₂₋₃ alkyl; or methylthiomethyl; R² is hydrogen C₁₋₄ ; alkyl;phenyl; phenyl mono-substituted with a member selected from the groupconsisting of methyl, trifluoromethyl, hydroxy, methoxy, fluoro, chloro,bromo, and iodo; or indolyl; R³ is C₃₋₆ alkyl; C₃₋₇ cycloalkyl; phenyl;or phenyl mono-substituted with a member selected from the groupconsisting of methyl, trifluoromethyl, hydroxy, methoxy, fluoro, chloro,bromo, and iodo; R⁴ is hydrogen; or ##STR29## where R' is hydrogen; C₁₋₄alkyl; hydroxy; or C₃₋₇ cycloalkyl R⁵ is 4-imidazolyl; amino C₂₋₄ alkyl;2-, 3-, or 4-pyridyl; or guanidyl C₂₋₃ alkyl; and E is OR", NHR", orN(R")₂, where each R" may be the same or different and is hydrogen orC₁₋₄ alkyl;wherein all of the asymmetric carbon atoms have an Sconfiguration, except those of substituents B and D, which may also havethe R configuration; and a pharmaceutically acceptable salt thereof. 10.A pharmaceutical composition for treating renin-associatedhyperaldosteronism, comprising a pharmaceutical carrier and atherapeutically effective amount of a peptide of the formula ##STR30##wherein: A is hydrogen; ##STR31## where n is 0 to 5 and R³ has the samemeaning as set out further below, and may additionally be hydrogen;B isabsent; glycyl; sarcosyl; or ##STR32## D is absent; or ##STR33## where Zis (CH₂)_(n) and n is 1 or 2; or --S--; R¹ is hydrogen; C₁₋₄ alkyl;hydroxy C₁₋₄ alkyl; phenyl; phenyl mono-substituted with a memberselected from the group consisting of methyl, trifluoromethyl, hydroxy,methoxy, fluoro, chloro, bromo, and iodo; indolyl; 4-imidazolyl; amineC₂₋₄ alkyl; guanidyl C₂₋₃ alkyl; or methylthiomethyl; R² is hydrogenC₁₋₄ ; alkyl; phenyl; phenyl mono-substituted with a member selectedfrom the group consisting of methyl, trifluoromethyl, hydroxy, methoxy,fluoro, chloro, bromo, and iodo; or indolyl; R³ is C₃₋₆ alkyl; C₃₋₇cycloalkyl; phenyl; or phenyl mono-substituted with a member selectedfrom the group consisting of methyl, trifluoromethyl, hydroxy, methoxy,fluoro, chloro, bromo, and iodo; R⁴ is hydrogen; or ##STR34## where R'is hydrogen; C₁₋₄ alkyl; hydroxy; or C₃₋₇ cycloalkyl; R⁵ is4-imidazolyl; amino C₂₋₄ alkyl; 2- , 3-, or 4-pyridyl; or guanidyl C₂₋₃alkyl; and E is OR"; NHR", or N(R")₂, where each R" may be the same ordifferent and is hydrogen or C₁₋₄ alkyl;wherein all of the asymmetriccarbon atoms have an S configuration, except those of substituents B andD, which may also have the R configuration; and a pharmaceuticallyacceptable salt thereof.
 11. A method of treating renin-associatedhyperaldosteronism, comprising administering to a patient in need ofsuch treatment, a therapeutically effective amount of a peptide of theformula: ##STR35## wherein: A is hydrogen; ##STR36## where n is 0 to 5and R³ has the same meaning as set out further below, and mayadditionally be hydrogen;B is absent; glycyl; sarcosyl; or ##STR37## Dis absent; or ##STR38## where Z is (CH₂)_(n) and n is 1 or 2; or --S--;R¹ is hydrogen; C₁₋₄ alkyl; hydroxy C₁₋₄ alkyl; phenyl; phenylmono-substituted with a member selected from the group consisting ofmethyl, trifluoromethyl, hydroxy, methoxy, fluoro, chloro, bromo, andiodo; indolyl; 4-imidazolyl; amine C₂₋₄ alkyl; guanidyl C₂₋₃ alkyl; ormethylthiomethyl; R² is hydrogen C₁₋₄ ; alkyl; phenyl; phenylmono-substituted with a member selected from the group consisting ofmethyl, trifluoromethyl, hydroxy, methoxy, fluoro, chloro, bromo, andiodo; or indolyl; R³ is C₃₋₆ alkyl; C₃₋₇ cycloalkyl; phenyl; or phenylmono-substituted with a member selected from the group consisting ofmethyl, trifluoromethyl, hydroxy, methoxy, fluoro, chloro, bromo, andiodo; R⁴ is hydrogen; or ##STR39## where R" is hydrogen; C₁₋₄ alkyl;hydroxy; or C₃₋₇ cycloalkyl; R⁵ is 4-imidazolyl; amino C₂₋₄ alkyl; 2-,3-, or 4-pyridyl; or guanidyl C₂₋₃ alkyl; and E is OR"; NHR", or N(R")₂,where each R" may be the same or different and is hydrogen or C₁₋₄alkyl;wherein all of the asymmetric carbon atoms have an Sconfiguration, except those of substituents B and D, which may also havethe R configuration; and a pharmaceutically acceptable salt thereof. 12.A method of determining the presence of renin-associated hypertension ina patient, comprising administering to such a patient, at a hypotensivedosage level and as a single dose, a peptide of the formula: ##STR40##wherein: A is hydrogen; ##STR41## where n is 0 to 5 and R³ has the samemeaning as set out further below, and may additionally be hydrogen;B isabsent; glycyl; sarcosyl; or ##STR42## D is absent; or ##STR43## where Zis (CH₂)_(n) and n is 1 or 2; or --S--; R¹ is hydrogen; C₁₋₄ alkyl;hydroxy C₁₋₄ alkyl; phenyl; phenyl mono-substituted with a memberselected from the group consisting of methyl, trifluoromethyl, hydroxy,methoxy, fluoro, chloro, bromo, and iodo; indolyl; 4-imidazolyl; amineC₂₋₄ alkyl; guanidyl C₂₋₃ alkyl; or methylthiomethyl; R² is hydrogenC₁₋₄ ; alkyl; phenyl; phenyl mono-substituted with a member selectedfrom the group consisting of methyl, trifluoromethyl, hydroxy, methoxy,fluoro, chloro, bromo, and iodo; or indolyl; R³ is C₃₋₆ alkyl; C₃₋₇cycloalkyl; phenyl; or phenyl mono-substituted with a member selectedfrom the group consisting of methyl, trifluoromethyl, hydroxy, methoxy,fluoro, chloro, bromo, and iodo; R⁴ is hydrogen; or ##STR44## where R'is hydrogen; C₁₋₄ alkyl; hydroxy; or C₃₋₇ cycloalkyl; R⁵ is4-imidazolyl; amino C₂₋₄ alkyl; 2-, 3- , or 4-pyridyl; or guanidyl C₂₋₃alkyl; and E is OR"; NHR", or N(R")₂, where each R" may be the same ordifferent and is hydrogen or C₁₋₄ alkyl;wherein all of the asymmetriccarbon atoms have an S configuration, except those of substituents B andD, which may also have the R configuration; and a pharmaceuticallyacceptable salt thereof; followed by monitoring of said patient's bloodpressure.